Cell-free extracts of Mvcoplasma pneumoniae showed two distinct reduced nicotinamide adenine dinucleotide (NADH2) oxidase activities in the supernatant fraction. By ammonium sulfate fractionation and polyacrylamide gel electrophoresis, one activity not requiring flavine co-factors was precipitated by 50 to 70% ammonium sulfate concentration and identified with a slower-moving band on acrylamide gel electrophoresis; a second NADH2 oxidase activity was flavine mononucleotide (FMN) dependent and associated with a more rapidly moving band; it could only be partially precipitated by ammonium sulfate concentrations ranging from 50 to 100%. Studies with alternate electron acceptors indicated the presence of a menadione, a 2,6-dichlorophenol indophenol and a very weak ferricyanide oxido-reductase activity, but no cytochrome c oxido-reductase, in the cell-free preparations. The NADH2 oxidase activities of all fractions were relatively cyanide insensitive and were only minimally inhibited by flavoprotein and other respiratory chain inhibitors. H202 formation was negligible unless FMN, but not flavine adenine dinucleotide (FAD), was added to the crude NADH2 oxidase system; upon fractionation and electrophoresis, the H202 formation was associated with the FMN-dependent, more rapidly moving NADH2 oxidase band. This FMN-dependent NADH2 oxidase-H202 generating system may be a mechanism for the H202 formation observed during glucose oxidation in the intact organism. H202 has long been implicated in the pathogenicity of Mycoplasma pneumoniae for the human respiratory tract and cold agglutinin formation during infection. This peroxide has been identified by Somerson et al. (27) as the low-molecular-weight hemolysin that produces a diagnostic, rapid lysis of sheep or guinea pig red cells. However, the exact biochemical reaction or reactions in which H202 is formed have not been identified for either M. pneumoniae or any other H202-secreting mycoplasma. It seemed logical to examine cell-free extracts (CFE) of M. pneumoniae for reduced nicotinamide adenine dinucleotide (NADH2) and NADH phosphate (NADPH2) oxidase activity and especially the very active soluble NADH2 oxidase as a possible source of H202 formation. MATERIALS AND METHODSOrganisms, media, and cultivation. Exponentialphase organisms of the FH and Mac strains of M. pneumoniae were grown in Hayflick medium, washed once, and concentrated as 10% cell suspensions as previously described (19, 20). CFE. The concentrated cell suspensions of 4 x 109 colony-forming units per ml in 0.0067 M sodiumpotassium phosphate buffer (pH 7.0) were disrupted in early experiments by 10 min of sonic oscillation with a 10-kc Raytheon oscillator at 100 to 150 mA; more recent CFE preparations were obtained with a Branson W 185 sonic oscillator at a 20-kHz frequency and power output at step 5 for five 1-min periods, alternating with